Title: Western Region Hazardous Substance Research Center Project 2-OSU-06
Development and Evaluation of Field Sensors for Monitoring Anaerobic Dehalogenation after Bioaugmentation for In-Situ Treatment of PCE and TCE

Investigator: James D. Ingle, Jr.; Oregon State University

Institution: Oregon State University

Research Category: Dehalogenation, bioaugmentation, PCE, groundwater

Project Period: 2004-2007


Goals: The purpose of this study was to develop, refine, and use sensors and field instruments, based on redox indicators and other reagents as on-site, on-line, or in-situ monitoring tools for assessing and optimizing redox and related conditions for treatment of PCE and TCE with dehalogenating and other cultures. These sensors and field instruments were calibrated for evaluating redox conditions and the effectiveness of dechlorination in collaborative situations involving a bioaugmentation approach in packed sediment columns.

Rationale: Better field and portable monitoring techniques for redox status and related conditions for bioremediation are needed 1) for the evaluation of laboratory samples, models such as columns and PAMs, and subsurface conditions at a site, 2) for continued assessment of the progress of remediation, and 3) for examination of the effects of bioaugmentation in field and laboratory experiments. We have demonstrated that redox sensors based on redox indicators exhibit promise for monitoring environmental redox levels. Research is needed to 1) identify and compare the response of these indicators during bioaugmentation, 2) improve the monitoring devices and methodology (flow cells, fiber optic probes, sampling) for practical use, 3) demonstrate that these devices and methodology can be employed for on-line or in situ monitoring of the status of anaerobic dehalogenating cultures in laboratory systems, and 4) develop new sensing species, methods, instrumental components and sensor designs for on-line monitoring of the status of dechlorinating and other anaerobic systems in columns and PAMs packed with soil, microcosm bottles, and sub-surface systems in the field.

Approach: Redox indicators immobilized on transparent, polymer films have been shown to be able to differentiate between different microbial redox levels and to predict whether conditions are appropriate for reductive dechlorination to occur. These redox indicators, which are incorporated into specially constructed flow sensors and fiber optic probes, were deployed in collaborative experiments for calibration and demonstration of their applicability. These experiments involved continuous monitoring of the redox conditions of cultures inside columns and PAMs packed with soil and enriched with halorespiratory cultures as a tool for spatial monitoring of dechlorination and to improve conditions necessary for effective dechlorination of PCE and TCE. In addition, we sought to investigate alternative sampling/reagent/detection systems, quantitative measurement of concentrations of reductants, O2, and fiber optic sensors.

Status: We improved portable, flow-based monitoring systems based on measuring the absorbance of immobilized redox indicators. The design and characteristics of the redox sensor monitoring systems were significantly modified to minimize oxygen permeation (contamination) and provide portability for easy operation in the lab and field. The flow sensors were used to successfully examine redox conditions in microcosm bottles containing a dechlorinating culture (Evanite culture) and in packed columns augmented with the culture. Consistently, we demonstrated that the rapid reduction of the indicator immobilized thionine (THI) indicated that conditions were appropriate for dechlorination and occurred with hours of contact with an active dechlorinating culture. The redox indicator cresyl violet (CV) was slowly but consistently reduced (about 25 to 75% reduced) as the culture became more reducing, typically during the dechlorination of cis-DCE and VC. It appears likely that reduced species other than S(-II) or Fe(II) contributed to the reduction of the indicators and preliminary results suggested some of these reductants may be products of cellular energy generation, mediators, or co-factors.

We constructed a fiber optic redox probe with immobilized redox indicator film at its tip and used it to monitor redox status by measuring the indicator absorbance providing in situ information about the redox conditions in the center of the columns in two laboratories. The fiber optic redox probe and flow redox sensor were installed in the same column and shown to respond comparably to the same redox conditions.

A new method was developed for determining reductive capacity (RC) with redox indicators. Based on a relatively uncomplicated concept, namely through determination of the total number of moles of indicator that react with an anaerobic sample, the concentration of reductants in an anaerobic sample is determined. This novel technique provides a new tool to evaluate redox status of anoxic and anaerobic samples in laboratory and field studies. RC provides information that is different from the “redox level” sensed by immobilized indicators. An immobilized indicator may be totally reduced, but the RC for the same indicator could still be increasing if microbial activity increases and concentrations of reductants increase in the sample.

In microcosm bottles inoculated with an active EV culture, RC (THI) ranged from about 100-400 µM and increased as the culture dechlorinated PCE to ETH. A large relative drop in RC likely suggests that the concentration of a critical species such as an electron donor has dropped and significantly impaired microbial activity or that the culture may have been compromised by contamination with O2. Data suggest that measurements of RC likely probe reductants that are associated with outer cell membranes or within cells. RC values are much greater than S(-II) or Fe(II) concentrations and the measured RC drops considerably when solutions obtained from a microcosm bottle are filtered. Reductive capacities were also measured in packed columns where the overall RC(THI) observed was lower (100-200 µM), which suggests that some of the RC measured in free cultures was not present due to cells or particles becoming trapped or attached in the column. Upon filtration of column samples, RC(THI) decreased by ~50%, which suggests that a larger fraction of the RC was due to reductants in solution compared to cultures in microcosm bottles.

We have a simple system to determine very low oxygen levels in laboratory samples and groundwater in the field. It is based on measuring the increase in absorbance of the redox indicator indigo carmine at 610 nm. First, 2 mL of the indigo carmine that is pre-reduced with H2 and a Pt catalyst is added to the a spectrometer sample cell and then 0.5 mL of the sample is added with a manual syringe or an automated miniature syringe pump. The method provides detection of O2 at levels below 1 ppm which cannot be accurately measured with a DO probe and the detection limit is 0.04 ppm. All components in the sample transfer system and sample cell were optimized to minimize O2 contamination. Several novel innovations were incorporated.

Anaerobic cultures were grown in septum bottles and were tested for DO. Microcosms dominated by Fe(III)-reducing, sulfate reducing or methanogenic conditions were prepared by seeding appropriate media with fresh wastewater sludge. As expected, none of the sulfate reducing and methanogenic cultures had detectable DO concentrations (above 0.04 mg/L). Interference of Fe(III) was observed in Fe(III)-reducing cultures possibly due to excess amount of iron hydroxide colloids.

We have constructed a versatile, integrated, microfluidic analysis platform fabricated using standard photolithography techniques and polydimethylsiloxane (PDMS) replica molding. This platform is designed for laboratory and field analysis of species (e.g., redox-active species such as S(-II) or Fe(II)) based on mixing a sample with a reagent and measuring the absorbance of the colored product formed. Various fluidic components have been developed individually and then integrated on one platform. These components include lateral percolation filters of different post sizes, micromixers that utilize lamination and geometric focusing to reduce diffusion distances, and a 1-cm long micro-flow cell with integrated fiber optics for spectroscopic detection. The small size of the platform (centimeters) and fluid channels of dimensions 50 to 200 mm make quite portable and suitable for long term monitoring because flow rate and sample and reagent consumption are very small (i.e., microliters).

We have successfully developed and evaluated the majority of the fluidic components necessary to in the future make an integrated microfluidic system that can be lowered into wells for chemical analysis. A microfabricated filter has been developed and characterized with the intent of allowing raw sample reagent to be pumped into the microfluidic device without pre-treatment. The filter was also used with a real environmental sample, Willamette River water, to demonstrate the application of pretreatment in environmental monitoring. A microfluidic reactor capable of mixing a sample and a reagent (or reagent mixture) by diffusion has been fabricated and the mixing efficiency has been determined in terms of the length needed for a given continuous flow rate. The fluid channels have dimensions 50 to 200 mm. An optical flow cell for performing UV/Visible spectrometry was developed, coupled to optical fibers implanted into the microfluidic chip, and evaluated a with miniaturized Ocean Optics light source and CCD array detector.

A method for packaging about 1 mL of reagent into a heat-sealed, collapsible reagent bag has been developed. A novel micropump (peristaltic) prototype that also provides valve functionality has been constructed and characterized and provides a relatively constant flow rate in the range of 1 to 80 μL/min. The pump requires low power to operate. Miniature light sources, detectors, and specific electronics components (microprocessors, pump driver circuitry, power management, miniature power supplies, etc.) have been purchased and evaluated



Journal Articles

Jones, B. D. and Ingle, Jr., J.D. (2005). Evaluation of redox indicators for determining sulfate-reducing and dechlorinating conditions. Water Research, 39, 4343-4350.

Koch, Corey R., James D. Ingle, Vincent T. Remcho. Bonding Upchurch NanoPorts to PDMS. Lab on a Chip (submitted 2007).

Koch, Corey R., James D. Ingle, Vincent T. Remcho. Magnets for facile molding of via holes in PDMS. Lab on a Chip (submitted 2007).

Ruiz-Haas, P. and Ingle, J.D, Jr. (2007). Monitoring Redox Conditions with Flow-Based and Fiber Optic Sensors Based on Redox Indicators: Application to Reductive Dehalogenation in a Bioaugmented Soil Column. Geomicrobiology Journal, Vol. 24 (3/4) 365-378.

Ruiz-Haas, P. and Ingle, J. D, Jr.. Monitoring of Redox State in a Dechlorinating Culture with Redox Indicators. Journal of Environmental Monitoring (submitted October 2007).


Abstracts and Posters

Cakin, Defne, and J.D. Ingle, Jr. (2004). Design and Characterization of a Liquid Core Waveguide based Analytical Device for the Analysis of Anaerobic Systems. EPA/ORD/HSRC Superfund Research on Risk Characterization and Monitoring, Las Vegas, NV.

Cakin, Defne, and James Ingle Jr. (2007). Methodology of Low Volume Sampling for Monitoring Oxygen and Reduced Species in Anaerobic Cultures. Subsurface Biosphere Initiative Workshop/IGERT Retreat, Newport, OR (June).

Koch, Corey R.,Vincent T. Remcho, and James D. Ingle, Jr. (2007). Microfluidics in the Subsurface: towards in-situ analysis of microliter volumes in a miniaturized package. Subsurface Biosphere Initiative Workshop/IGERT Retreat, Newport, OR (June).

Ruiz-Haas, Peter and J.D. Ingle, Jr. (2004). Evaluation of Redox Conditions with Redox-Indicator Based Sensors in Soil and Microcosms Bioaugmented with Reductive Dehalogenating Bacteria. EPA/ORD/HSRC Superfund Research on Risk Characterization and Monitoring, Las Vegas, NV

Ruiz-Haas, Peter, and J.D. Ingle, Jr. (2005). Monitoring of Redox Conditions with Redox-Indicator Based Sensors in Soil Columns and Microcosms Bioaugmented with Reductive Dehalogenating Bacteria. Joint International Symposia for Subsurface Microbiology and Environmental Biogeochemistry, Jackson, WY; and American Chemical Society (ACS) Fall National Meeting, Washington, DC.


Ruiz-Haas, P. (2006). Monitoring Redox Conditions with Redox Indicators during Microbial Reductive Dechlorination in Microcosms and Bioaugmented Columns, Ph.D., Oregon State University.

Supplemental Keywords: biotransformation; characterization; VOCs; chlorinated solvents; bioremediation; environmental chemistry